Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. Acute insults to the nervous system, such as stroke or traumatic brain injury, cause an increase in extracellular glutamate, excessive activation of glutamate receptors, and neuronal death through a process called excitotoxicity. Excitatory synaptic transmission is also an energy consuming process. In fact, increases in excitatory activity cause an increase in blood flow to meet energetic demands imposed by this excitatory activity. Compared to most other neurotransmitters, glutamate is relatively uniquely cleared into astrocytes rather than being directly recycled back into the nerve terminal. Two Na+-dependent glutamate transporters, GLT-1 and GLAST (also called EAAT2 and EAAT1), are almost exclusively expressed by astrocytes. In astrocytes, expression of GLT-1 and GLAST is enriched on fine processes near synapses. During our first funding cycle, we studied the co-compartmentalization of GLT-1 and GLAST with mitochondria. We demonstrated mitochondria are found throughout these processes, they are mobile, and the percentage of mobile mitochondria is regulated by neuronal activity. Furthermore, we demonstrated that inhibition of glutamate transport or inhibition of reversed operation of the Na+/Ca2+ exchanger increases the percentage of mobile mitochondria; we showed that these effects are accompanied by a decrease in basal Ca2+ in astrocyte processes. We developed several lines of evidence that strongly suggest that mitochondria shape spontaneous Ca2+ spikes (amplitude, duration, and spread) in astrocyte processes. We showed that oxygen glucose deprivation causes a loss of mitochondria from astrocytic processes. We showed that inhibition of glutamate transport or inhibition of the reversed operation of the Na+/Ca2+ exchanger blocks this loss of mitochondria. Our data suggest that the elevations in extracellular glutamate observed with acute insults, such as stroke, cause a loss of astrocytic mitochondria. The mechanism by which glutamate transporters cause this loss of mitochondria has not been defined, and it is not clear if this loss has pathologic consequences. In this renewal, we will define the mechanisms involved in this loss of mitochondria and determine if this loss contributes to the pathologic consequences of stroke. We will also determine if glial glutamate transport, reversed Na+/Ca2+ exchange, and mitochondria control the increase in blood flow observed with excitatory neuronal activity.